In specialized secretory cells that produce and release biologically active substances in a regulated fashion, tight control of both the quantity and quality of secretory material is of paramount importance. During crinophagy, abnormal, excess or obsolete secretory granules directly fuse with lysosomes to yield crinosomes, in which the delivered secretory material is degraded. Crinophagy maintains the proper intracellular pool of secretory granules, and it is enhanced when secretory material accumulates because of compromised secretion. Recent studies highlight that it can even degrade newly formed, nascent secretory granules that shed from the trans-Golgi network. This implies that crinophagy provides a quality control checkpoint acting at the formation of secretory vesicles, and this degradation mechanism might survey secretory granules throughout their maturation. Of note, a plethora of human disorders is associated with defective lysosomal clearance of secretory material via crinophagy or similar pathways, including macro- or micro-autophagic degradation of secretory granules (referred to here as macro- and micro-secretophagy, respectively). In our Review, we summarize key recent advances in this field and discuss potential links with disease.

Eukaryotic cells can target proteins to the extracellular space, cell surface or lysosomes through transport via the secretory pathway. Small secretory vesicles (SVs) rapidly deliver constitutively secreted proteins to the cell surface, where stimulus-independent discharge of contents takes place. In contrast, specialized endocrine, neuroendocrine and exocrine secretory cells store highly condensed cargoes (such as hormones and neuropeptides) in secretory granules (SGs), which are usually larger than SVs, until their regulated release is triggered by a secretagogue (Burgess and Kelly, 1987; Wendler et al., 2001). The proper sorting and packaging of secretory cargoes in the trans-Golgi network (TGN), as well as the formation, maturation and release of SGs by exocytosis, are tightly controlled processes. The coordinated action of several regulatory proteins ensures the fidelity of protein transport along the entire regulated secretory pathway (RSP), and these regulatory proteins include sorting receptors, coat proteins with their adaptors, small GTPases, tethering factors and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), among others. Autophagy is a highly conserved pathway for regulated lysosomal clearance of cellular components, including secretory material. As will be discussed in more detail below, SGs or their contents are transported to lysosomes via different routes in secretory cells: macroautophagy, microautophagy, and crinophagy. During crinophagy, SGs directly fuse with lysosomes to form degradative crinosomes (Fig. 1). This process was first observed in the 1960s in lactotroph hormone (LTH)-producing cells of the anterior pituitary gland upon inhibition of secretion (Smith and Farquhar, 1966). The simplicity of this experimental setup was amazing: by weaning suckling rat pups from their mothers, it was possible to precisely manipulate (that is, switch off) secretion and thus trigger crinophagy without using modern genetic tools, which were not yet available (Smith and Farquhar, 1966). Similarly, lysosomal degradation of the secretory material was later described in pancreatic β-cells using both morphological (Creutzfeldt et al., 1969) and biochemical analyses (Halban and Wollheim, 1980). Crinophagy has subsequently been observed in various other secretory cell types, such as hepatocytes (Glaumann, 1989; Glaumann et al., 1982), pancreatic acinar (Kitagawa and Ono, 1986; Koike et al., 1982) and α-cells (Orci et al., 1968), thyroid and parathyroid cells (Gilloteaux and Pardhan, 2015), and ameloblasts (Nanci et al., 1985). In the salivary gland of last (L3)-stage Drosophila larvae, a low pulse of 20-hydroxyecdysone (20E) induces the production of highly glycosylated secretory granule secretion (Sgs) proteins, which are packaged into mucous SGs (so-called glue granules) (Biyasheva et al., 2001). Their release is triggered by a higher pulse of 20E, and after the secreted glue leaves the lumen of salivary glands it adheres the forming (pre)pupa to a solid surface for the duration of metamorphosis (Costantino et al., 2008). Drosophila larval salivary gland cells have proven to be a useful model to study regulated secretion and crinophagy (Boda et al., 2019; Burgess et al., 2011; Csizmadia et al., 2022, 2018; Torres et al., 2014) (Fig. 2).

Fig. 1.

Coordination of SG maturation and crinophagy. Non-regulated secretory cargo is transported by constitutive SVs, whereas regulated secretory proteins are sorted at the TGN into forming nascent iSGs (sorting for entry), which can either continue their maturation or be degraded by crinophagy (red arrows). Non-granular components, including lysosomal hydrolases, are sorted into the forming SGs due to their bulk transport across the TGN. They are subsequently re-sorted (sorting for exit) via CCVs (depicted with their clathrin coat indicated) from iSGs to reach their destinations (TGN, plasma membrane or lysosomes, purple arrows). SG maturation is highly complex and involves the homotypic fusion of iSGs, an intermediate endosomal sorting station and extensive material exchange between endosomes, maturing SGs and the TGN (green arrows). Mature SGs (with retained material) are stored in the cytoplasm until their rapid release is triggered by secretion-promoting stimuli, or they can also be degraded by crinophagy (blue arrows). Old or abnormal SGs (with improper content and/or membrane composition) are thought to be more prone to crinophagy, whereas younger SGs are preferentially released by cells. AP2, adaptor protein 2 complex; SytIV, synaptotagmin IV.

Fig. 1.

Coordination of SG maturation and crinophagy. Non-regulated secretory cargo is transported by constitutive SVs, whereas regulated secretory proteins are sorted at the TGN into forming nascent iSGs (sorting for entry), which can either continue their maturation or be degraded by crinophagy (red arrows). Non-granular components, including lysosomal hydrolases, are sorted into the forming SGs due to their bulk transport across the TGN. They are subsequently re-sorted (sorting for exit) via CCVs (depicted with their clathrin coat indicated) from iSGs to reach their destinations (TGN, plasma membrane or lysosomes, purple arrows). SG maturation is highly complex and involves the homotypic fusion of iSGs, an intermediate endosomal sorting station and extensive material exchange between endosomes, maturing SGs and the TGN (green arrows). Mature SGs (with retained material) are stored in the cytoplasm until their rapid release is triggered by secretion-promoting stimuli, or they can also be degraded by crinophagy (blue arrows). Old or abnormal SGs (with improper content and/or membrane composition) are thought to be more prone to crinophagy, whereas younger SGs are preferentially released by cells. AP2, adaptor protein 2 complex; SytIV, synaptotagmin IV.

Fig. 2.

The maturation and crinophagy of Drosophila glue granules. (A) In early L3-stage Drosophila larvae, Sgs glycoproteins are packaged into smaller iSGs. (B) These iSGs then fuse with each other to form larger maturing dense-core secretory granules (mSG). (C) The content of the granules is then extensively rearranged to prepare them for secretion, which takes place at end of the L3 stage. (D) Persisting, non-released or abnormal granules can directly fuse with lysosomes to yield crinosomes, in which the granular components are degraded in prepupae. Scale bars: 1 µm. (E) Schematic representation of Drosophila glue granule maturation and degradation by crinophagy, which progresses similarly to that in mammals (see Fig. 1). At the TGN, Arl1 recruits the clathrin adaptor AP1 to promote the formation of iSGs. The iSGs then fuse with each other in a SNAP24-dependent manner. Retrograde trafficking from endosomes also contributes to the maturation and size increase of glue SGs. The mSG content is then extensively reorganized by ion uptake and progressive acidification to prepare the granules for their subsequent secretion. Residual, non-secreted glue granules fuse with multiple lysosomes to produce crinosomes. See Fig. 4 for factors involved in the crinophagic fusion step.

Fig. 2.

The maturation and crinophagy of Drosophila glue granules. (A) In early L3-stage Drosophila larvae, Sgs glycoproteins are packaged into smaller iSGs. (B) These iSGs then fuse with each other to form larger maturing dense-core secretory granules (mSG). (C) The content of the granules is then extensively rearranged to prepare them for secretion, which takes place at end of the L3 stage. (D) Persisting, non-released or abnormal granules can directly fuse with lysosomes to yield crinosomes, in which the granular components are degraded in prepupae. Scale bars: 1 µm. (E) Schematic representation of Drosophila glue granule maturation and degradation by crinophagy, which progresses similarly to that in mammals (see Fig. 1). At the TGN, Arl1 recruits the clathrin adaptor AP1 to promote the formation of iSGs. The iSGs then fuse with each other in a SNAP24-dependent manner. Retrograde trafficking from endosomes also contributes to the maturation and size increase of glue SGs. The mSG content is then extensively reorganized by ion uptake and progressive acidification to prepare the granules for their subsequent secretion. Residual, non-secreted glue granules fuse with multiple lysosomes to produce crinosomes. See Fig. 4 for factors involved in the crinophagic fusion step.

The main role of crinophagy might be to shape the stored pool of mature SGs (mSGs), as it clears damaged or obsolete SGs through its basal activity and is enhanced when the biosynthesis of cargo exceeds the secretory capacity of cells (Halban and Wollheim, 1980; Kamijo et al., 1986; Schnell et al., 1988) or upon compromised secretion (Antakly et al., 1979; Glaumann et al., 1982; Koike et al., 1982; Smith and Farquhar, 1966). Recent studies have reported crinophagic degradation of newly formed immature SGs (iSGs) (Csizmadia et al., 2022; Goginashvili et al., 2015; Li et al., 2022; Pasquier et al., 2019; Rajak et al., 2021; Zhou et al., 2020), suggesting that crinophagy can immediately eliminate abnormal vesicles. Thus, it appears to survey the entire formation and maturation processes of SGs as a quality controller. Furthermore, nascent granule degradation can also be induced in response to starvation to provide nutrients (Goginashvili et al., 2015; Rajak et al., 2021). Accumulating evidence points to a crucial role of crinophagy affecting secretory cells in several diseases, including type II diabetes (Li et al., 2022; Marsh et al., 2007; Pasquier et al., 2019), acute pancreatitis (Mareninova et al., 2015), Crohn's disease (Thachil et al., 2012), endocrine tumors (Weckman et al., 2015) and, presumably, endocrine hypo- and hyper-activities (Weckman et al., 2014). There is thus increasing demand for elucidation of the molecular basis of crinophagy and development of targeted therapeutic approaches that might help to treat these diseases (Box 1). Indeed, studies on pancreatic α- and β-cells and on larval salivary gland cells of Drosophila have already revealed some key molecular determinants of crinophagy (Kuriakose et al., 1989; Rajak et al., 2021; Sandberg and Borg, 2006, 2007) and the actual fusion event between SGs and lysosomes (Boda et al., 2019; Csizmadia et al., 2018; Li et al., 2022; Lőrincz et al., 2019; Zhou et al., 2020). In this Review, we briefly describe SG maturation and autophagic degradation routes in secretory cells. We then discuss how iSGs and mSGs can be degraded by autophagy (focusing on crinophagy), highlighting the role of this degradation in the quality control of SGs and presenting the known molecular mechanisms.

Box 1. The therapeutic potential of pharmacological targeting of crinophagy

Many endocrine and metabolic diseases are associated with either an excess of SGs, as occurs in endocrine hyperfunctions (Weckman et al., 2014), or a deficiency in SGs, as occurs in endocrine hypofunctions (Weckman et al., 2014), type 2 diabetes (Li et al., 2022; Pasquier et al., 2019) and Crohn's disease (Thachil et al., 2012). Pharmacological treatments specifically targeting crinophagy could allow efficient restoration of adequate SG levels without perturbing basal and other selective autophagic pathways. Recently, some molecular details of crinophagic degradation of iSGs and mSGs (Boda et al., 2023; Csizmadia et al., 2022; Li et al., 2022; Zhou et al., 2020), and its regulation by mTORC1 (Rajak et al., 2021) and steroid hormones (Sandberg and Borg, 2007) have been revealed in fly and human secretory cells, respectively. In the future, the universality of these factors should be investigated across different secretory cells and species. This would pave the way for the development of substances that target crinophagy-specific fusion factors or their interactions. Of note, some drugs are already being developed to selectively inhibit autophagosome–lysosome fusion. For instance, ethyl (2-(5-nitrothiophene-2-carboxamido)thiophene-3-carbonyl) carbamate (EACC) interferes with autophagosomal localization of STX17 and inhibits its interaction with the HOPS complex and VAMP8 (Vats and Manjithaya, 2019), whereas another inhibitor, berbamine, disrupts the interaction of SNAP29 with VAMP8 (Fu et al., 2018).

The RSP and crinophagy have been extensively studied in pancreatic β-cells, so here we aim to overview the main steps and regulation of the RSP by mainly focusing on β-granules (Fig. 1). Insulin is synthesized as preproinsulin, which is cleaved by signal peptidase in the endoplasmic reticulum (ER). The resulting proinsulin forms a zinc-stabilized hexamer (Emdin et al., 1980) and is transported towards the Golgi. Entering the TGN, proinsulin is sorted (‘sorting for entry’) via interactions with sorting receptors and lipid rafts (Tooze et al., 2001) and is packaged together with other granular components, such as prohormone convertases, chromogranin A (CHGA), chromogranin B (CHGB) and secretogranin II (SCG2) (Brunner et al., 2007; Natori and Huttner, 1996), into the forming SGs with 99% efficiency (Rhodes and Halban, 1987). Phosphatidylinositol 4-phosphate (PI4P) produced by phosphatidylinositol 4-kinase type IIα (PI4KIIα, also known as PI4K2A) and phosphatidylinositol 4-kinase type IIIβ (PI4KIIIβ, also known as PI4KB), and the ADP-ribosylation factor 1 (ARF1) have been shown to recruit clathrin to the surface of forming SG buds via the adaptor protein 1 complex (AP1) (Austin et al., 2000; Hausser et al., 2005; Wang et al., 2003). During SG loading, Bin-Amphiphysin-Rvs (BAR) domain-containing arfaptin 1 (ARFIP1), which is recruited by PI4P and ARF-like 1 (ARL1), can shield the neck of the forming vesicle from ARF1-mediated scission (Gehart et al., 2012; Man et al., 2011). When the vesicle reaches its proper size, protein kinase D1 (PRKD1) presumably phosphorylates and releases arfaptin 1, and the SG precursor detaches from the TGN (Gehart et al., 2012). The role of PRKD1 in transport vesicle fission and PI4P production via PI4KIIIβ phosphorylation has been demonstrated (Bossard et al., 2007; Hausser et al., 2005; Malhotra and Campelo, 2011), and p38δ mitogen-activated protein kinase (also known as MAPK13) can inhibit its activity (Sumara et al., 2009). SG biogenesis is also highly reliant on cholesterol transport, which is controlled by the coordinated action of ATP-binding cassette transporters ABCG1 and ABCA1, and oxysterol-binding protein (OSBP) (Hussain et al., 2018) (Fig. 1). Similarly, Drosophila glue granule biogenesis depends on AP1-mediated clathrin recruitment (Burgess et al., 2011) and Arl1 (Torres et al., 2014) (Fig. 2E).

Within nascent SGs, proinsulin is cleaved by prohormone convertases (PC1/3 and PC2, also known as PCSK1 and PCSK2, respectively) and carboxypeptidase E (CPE) to yield insulin and C-peptide (Arvan and Halban, 2004). The latter is cleared by constitutive-like secretion or rapidly degraded in a crinophagy-independent manner (Neerman-Arbez and Halban, 1993). Acidification of nascent SGs starts at the TGN and gradually increases (along with Ca2+ concentration) as the SGs mature to promote proinsulin processing (Davidson et al., 1988; Hummer et al., 2017; Orci et al., 1987). The mildly acidic milieu and elevated Ca2+ levels (Tooze et al., 2001; Yoo and Lewis, 1992) within iSGs also aid the aggregation of insulin and granin-family proteins (Natori and Huttner, 1996), which contributes to the formation of the dense core of SGs. HID-1 is involved in cargo sorting and dense-core formation through retaining vacuolar-type ATPase (V-ATPase) at the TGN (Hummer et al., 2017). V-ATPase might also have a role in protein sorting that is distinct from its function in acidification, as perturbation of V-ATPase activity gives rise to mixed vacuolar structures with secretory, lysosomal and autophagic components (Binger et al., 2019; Sobota et al., 2009). Aggregation makes insulin more resistant to lysosomal degradation (Halban et al., 1987) and is also crucial for its retention (‘sorting by retention’) within the RSP. Other missorted soluble non-granule components, such as lysosomal hydrolases, are recognized by mannose 6-phosphate receptors (M6PRs) and furin for selective removal (‘sorting for exit’) via clathrin-coated vesicles (CCVs) that are formed in an AP1-dependent manner (Dittié et al., 1999; Klumperman et al., 1998; Kuliawat and Arvan, 1994; Kuliawat et al., 1997; Molinete et al., 2001). CCVs with the retrieved material can be transported back to the TGN (as occurs for peptidylglycine α-amidating monooxygenase) or towards endo-lysosomal compartments (as occurs for syntaxin 6, VAMP4 and lysosomal proenzymes), or released by constitutive-like secretion (as in the case of C-peptide) (Dittié et al., 1999; Klumperman et al., 1998; Kuliawat and Arvan, 1994; Kuliawat et al., 1997; Li et al., 2022; Neerman-Arbez and Halban, 1993).

The final ∼250 nm size of mature β-granules (Fava et al., 2012) arises by homotypic fusion of iSGs. A pheochromocytoma (PC12) cell-derived in vitro iSG–iSG fusion assay has been used to demonstrate that the fusion of iSGs relies on the activity of N-ethylmaleimide-sensitive factor (NSF) and alpha-soluble NSF attachment protein (α-SNAP, also known as NAPA) (Urbé et al., 1998), the SNARE syntaxin 6 (STX6) (Wendler et al., 2001) and the Ca2+-sensor synaptotagmin IV (also known as SYT4; Ahras et al., 2006). In pancreatic β-cells, HID-1 regulates the homotypic fusion of immature insulin granules (Du et al., 2016) (Fig. 1). In Drosophila, glue granule maturation involves SNAP24-mediated homotypic fusion (Niemeyer and Schwarz, 2000) and a profound content reorganization (Fig. 2) mediated by progressive acidification and uptake of Cl and Ca2+ ions (Syed et al., 2022).

Recent studies on other secretory cell types support the importance of an endosomal contribution during proper maturation of SGs (Ma et al., 2021). In mast cells, RAB5 (herein referring to RAB5 isoforms in general) has been proposed to promote the fusion of Golgi-derived SGs with early endosomes to acquire endosomal cargoes such as CD63 (Azouz et al., 2022). The maturation of mucin-containing SGs of Drosophila salivary gland cells is similarly dependent on retrograde trafficking from early endosomes to the TGN. This is mediated by PI4KIIα, Past1, Syntaxin 16 (Syx16) and retromer (Fig. 2E), as well as other endosomal sorting, maturation or retrograde trafficking regulators (Ma et al., 2020; Neuman et al., 2021). In BON neuroendocrine and INS-1 insulinoma cell lines, the retrograde trafficking regulator BAIAP3 has been found to be necessary for proper dense-core vesicle (DCV) maturation. Golgi-localized Rab2 GTPase and its effector proteins RUN domain-containing protein (RUND-1) and CARD domain-containing protein 1 (CCCP-1) are required for proper sorting of soluble and transmembrane proteins into DCVs in Caenorhabditis elegans neuroendocrine cells; these are thus important regulators of DCV maturation (Ailion et al., 2014; Edwards et al., 2009). It is worth mentioning that a complex between EARP-interacting protein 1 (EIPR-1) and the endosome-associated recycling protein (EARP) complex acts in the same pathway as Rab2 (Topalidou et al., 2020, 2016). These results suggest that DCVs pass through an endosomal sorting compartment or undergo reciprocal transport with endosomes during their proper maturation (Topalidou et al., 2016) (Fig. 1).

PRKD1 and PI4P are key regulators of SG formation, which is followed by the complex process of SG maturation that involves processing, condensation and sorting of cargoes; SG membrane remodeling; acidification; ion uptake; and homotypic fusion of SGs. Maturation also relies on extensive transport between SGs and endosomes, as well as retrograde trafficking from endosomes to the TGN. As discussed below, disturbance of any of these processes can trigger the breakdown of secretory material by crinophagy.

Cells utilize constitutive basal autophagy to selectively remove superfluous or damaged constituents, such as protein aggregates or malfunctioning mitochondria, by delivering them to lysosomes where breakdown takes place (selective autophagy), and the released metabolites are then reused in anabolic processes. Starvation or rapamycin treatment induces bulk autophagy to provide nutrients for cell survival by en masse degradation of randomly sequestered material. Abnormal or excess SGs are subject to selective autophagic breakdown. Depending on how SGs reach lysosomes, their degradation can follow the pathways of macroautophagy, microautophagy and crinophagy. Based on the nomenclature used for other selective autophagy pathways, such as mitophagy and ERphagy, the former two processes can be referred to as macrosecretophagy and microsecretophagy, respectively (Fig. 3).

Fig. 3.

Main autophagic pathways for SG degradation. SGs in various secretory cells, such as pancreatic islet β-cells with SGs containing proinsulin and insulin, or pancreatic acinar cells with ZGs containing digestive enzymes, can be engulfed by autophagosomes and transported to lysosomes following a conventional pathway of selective macroautophagy (macrosecretophagy). The molecular mechanism of this pathway includes Atg proteins involved in canonical autophagosome formation, as well as selective receptors such as p62, recognizing potential SG ubiquitylation (Ub). Smaller SGs, such as insulin SGs, can also undergo selective microautophagy (microsecretophagy). In this case, they are engulfed by invaginations or protrusions of the lysosomal membrane (orange), but the molecular mechanisms of this process are mostly unknown. During crinophagy, SGs (including glucagon and insulin iSGs and mSGs, ZGs, neurohormone SGs and Drosophila glue granules) directly fuse with lysosomes to release their contents into the lysosomal lumen. This pathway enables the recycling of SG membrane (blue) components for further rounds of SG biogenesis. Of note, the size of SGs to be degraded by crinophagy is not limited. See Fig. 4 for details on the molecular mechanisms of crinophagy.

Fig. 3.

Main autophagic pathways for SG degradation. SGs in various secretory cells, such as pancreatic islet β-cells with SGs containing proinsulin and insulin, or pancreatic acinar cells with ZGs containing digestive enzymes, can be engulfed by autophagosomes and transported to lysosomes following a conventional pathway of selective macroautophagy (macrosecretophagy). The molecular mechanism of this pathway includes Atg proteins involved in canonical autophagosome formation, as well as selective receptors such as p62, recognizing potential SG ubiquitylation (Ub). Smaller SGs, such as insulin SGs, can also undergo selective microautophagy (microsecretophagy). In this case, they are engulfed by invaginations or protrusions of the lysosomal membrane (orange), but the molecular mechanisms of this process are mostly unknown. During crinophagy, SGs (including glucagon and insulin iSGs and mSGs, ZGs, neurohormone SGs and Drosophila glue granules) directly fuse with lysosomes to release their contents into the lysosomal lumen. This pathway enables the recycling of SG membrane (blue) components for further rounds of SG biogenesis. Of note, the size of SGs to be degraded by crinophagy is not limited. See Fig. 4 for details on the molecular mechanisms of crinophagy.

Fig. 4.

Factors involved in lysosomal fusions of SGs and other cargo. Fusion of Drosophila glue granules with lysosomes requires the coordinated action of Rab2, Rab7 and Arl8 small GTPases, the HOPS tethering complex, and a SNARE complex composed of Syntaxin 13, SNAP29 and VAMP7. Fusion factors involved in autophagosome–lysosome fusion are either the same (small GTPases) or similar (SNAREs): a complex comprising STX17, SNAP29 and either VAMP7 or VAMP8 (VAMP7/8) acts in parallel with the more recently identified STX7–SNAP29–YKT6 complex. Note that the crinophagic SNARE complex involves the Qa SNARE Syntaxin 13 instead of Syntaxin 17. Recently, RAB26 and the RAB7 effector RILP have been shown to promote crinophagy of immature insulin SGs in concert with a SNARE complex comprising STX7, STX8, VTI1B and VAMP4, resembling the late endosome–lysosome fusion machinery. The tethering factor(s) involved in iSG crinophagy have not yet been identified.

Fig. 4.

Factors involved in lysosomal fusions of SGs and other cargo. Fusion of Drosophila glue granules with lysosomes requires the coordinated action of Rab2, Rab7 and Arl8 small GTPases, the HOPS tethering complex, and a SNARE complex composed of Syntaxin 13, SNAP29 and VAMP7. Fusion factors involved in autophagosome–lysosome fusion are either the same (small GTPases) or similar (SNAREs): a complex comprising STX17, SNAP29 and either VAMP7 or VAMP8 (VAMP7/8) acts in parallel with the more recently identified STX7–SNAP29–YKT6 complex. Note that the crinophagic SNARE complex involves the Qa SNARE Syntaxin 13 instead of Syntaxin 17. Recently, RAB26 and the RAB7 effector RILP have been shown to promote crinophagy of immature insulin SGs in concert with a SNARE complex comprising STX7, STX8, VTI1B and VAMP4, resembling the late endosome–lysosome fusion machinery. The tethering factor(s) involved in iSG crinophagy have not yet been identified.

During macrosecretophagy, cup-shaped phagophores enclose part of the cytoplasm (including whole organelles – in this case SGs) to form double-membrane autophagosomes, which subsequently fuse with lysosomes to generate autolysosomes where cargo is degraded along with the inner autophagosomal membrane (Mizushima et al., 2011; Morishita and Mizushima, 2019; Nakatogawa, 2020). Macrosecretophagy can sequester both proinsulin-containing iSGs (Riahi et al., 2016) and insulin-containing mSGs (Marsh et al., 2007; Yamamoto et al., 2018) in pancreatic β-cells. The pancreatic acinar cells store the synthetized digestive enzymes in an inactive zymogen form in SGs called zymogen granules (ZGs). Trypsinogen can be activated prematurely in acute pancreatitis, resulting in tissue autodigestion and inflammation. Zymophagy (which we consider a type of macrosecretophagy) can selectively degrade these prematurely activated ZGs upon their ubiquitylation to promote cell survival (Grasso et al., 2011). Recently, trypsin ubiquitylation by the E3 ubiquitin ligase TRIM33 has been identified as the main regulator of this process (Wang et al., 2022), in addition to the ubiquitin protease USP9x (Grasso et al., 2011). More than 40 autophagy-related (Atg) proteins that regulate autophagosome formation have been identified in yeast and subsequently in other eukaryotes (Morishita and Mizushima, 2019). These can be grouped into the following functional units: the Atg1/ULK1 kinase complex, which is crucial for autophagy initiation and is inhibited by the upstream (mammalian) target of rapamycin complex 1 [(m)TORC1]; Atg9-family transmembrane proteins, which are involved in phagophore generation; a VPS34 (PIK3C3) phosphatidylinositol 3-kinase (PI3K) complex producing phosphatidylinositol 3-phosphate (PI3P); the Atg2–WIPI (WD-repeat protein interacting with phosphoinositides; yeast Atg18 homolog) complex, which binds to PI3P and is involved in phagophore expansion; and the ubiquitin-like Atg8 family (such as LC3 proteins, also known as MAP1LC3 proteins) and Atg12 proteins, with their conjugation enzymes such as Atg7. Phosphatidylethanolamine (PE)-conjugated LC3 plays a key role in selective SG engulfment as it interacts with autophagic receptors such as p62 (also known as sequestosome 1, SQSTM1) on the concave surface of phagophores (Mizushima et al., 2011; Nakatogawa, 2020).

During microsecretophagy, either invaginations or protrusions of the lysosomal membrane can wrap around and internalize SGs into the lumen of lysosomes (Marsh et al., 2007), where this part of the lysosomal membrane is also degraded (Oku and Sakai, 2018). During crinophagy, SGs directly fuse with lysosomes (Csizmadia et al., 2018; Goginashvili et al., 2015; Li et al., 2022; Pasquier et al., 2019; Smith and Farquhar, 1966), so in this case, the membrane components of SGs are not degraded and can be recycled (Marsh et al., 2007; Weckman et al., 2014), perhaps by autophagic lysosome reformation (Yu et al., 2010).

We note here that in addition to these pathways, regulated secretory proteins such as proinsulin can also be removed before leaving the ER by ERphagy (Chino and Mizushima, 2023) or by ER-associated degradation (ERAD) via the ubiquitin–proteasomal system before they are completely folded (Sugawara et al., 2014). Misfolded proteins are recognized by ER chaperones that facilitate retrotranslocation into the cytosol, where they are polyubiquitylated by E3 ubiquitin ligases to trigger proteasomal degradation (Vembar and Brodsky, 2008).

Metabolic stress, such as starvation or glucolipotoxicity, induces crinophagic breakdown of immature proinsulin granules within CD63-positive lysosomes (also known as stress-induced nascent granule degradation, SINGD), due to reduced expression of PRKD1 in pancreatic β-cells (Goginashvili et al., 2015; Pasquier et al., 2019). PRKD1 oppositely regulates the key steps of SG formation (see above) and SINGD, promoting biogenesis (Bossard et al., 2007; Gehart et al., 2012; Malhotra and Campelo, 2011) and inhibiting degradation (Goginashvili et al., 2015; Pasquier et al., 2019). Amino acids released during lysosomal degradation promote the recruitment of mTORC1 to the surface of lysosomes and its activation, leading to inhibition of autophagy and decreased insulin secretion (Goginashvili et al., 2015; Pasquier et al., 2019; Vivot et al., 2020). Macroautophagy stimulates insulin release by an as-yet-unknown mechanism, so SINGD plays a dual role in starvation by supplying necessary nutrients for cell survival, while keeping both macroautophagy and insulin secretion low (Goginashvili et al., 2015). Furthermore, SINGD activation might contribute to β-cell dysfunction during diabetes by interfering with protective basal autophagy and causing insulin deficiency (Pasquier et al., 2019). Cellular selenoproteins inhibit SINGD and ferroptosis-like cell death, thus selenium might be important for proper β-cell function and to prevent diabetes (Kitabayashi et al., 2022). In glucagon-producing pancreatic α-cells, the mTORC1 inhibitor rapamycin or amino acid shortage causes crinophagic degradation of the glucagon granules and induced autophagy to ensure nutrient replenishment (Rajak et al., 2021). Glucagon SG–lysosome fusion appears to be independent of macroautophagy, as it does not require Atg5 and Beclin-1 (also known as Atg6) (Rajak et al., 2021), similar to what has been found for SINGD in β-cells (Pasquier et al., 2019). However, differences regarding mTORC1 and autophagic activities (that is, in α-cells, blocking mTORC1 activates both conventional autophagy and crinophagy, whereas in β-cells, enhanced crinophagy leads to inhibition of autophagy via mTORC1 activation) remain unresolved. Knockdown of key determinants of SG biogenesis at the TGN, such as arfaptin 1 (Gehart et al., 2012) or the cholesterol regulators ABCG1 and OSBP (Hussain et al., 2018), has been reported to trigger early lysosomal degradation of nascent granules that presumably have altered membrane lipid composition, size or content. Similar phenotypes are seen when dense-core formation is prevented by either loss of HID-1 function (Hummer et al., 2017) or impairment of retrograde endosome-to-TGN routes (which contribute to SG maturation) following knockdown of BAIAP3 in INS-1 insulinoma cells (Zhang et al., 2017). In Drosophila salivary gland cells, silencing of the Golgi-associated retrograde protein (GARP) complex, Rab6 small GTPase, or the SNAREs Syx16 or Syb (Synaptobrevin), which are all involved in retrograde endosome-to-TGN transport, similarly leads to an accumulation of iSGs that are directed towards lysosomal degradation (Csizmadia et al., 2022). Thus, early activation of crinophagy can be triggered either by improperly formed SGs (perhaps to prevent clogging of the RSP) or by metabolic stress (see SINGD above). In autophagy-deficient β-cells, a compensatory process termed Golgi membrane-associated degradation (GOMED) is induced when PI4P-dependent SG biogenesis is perturbed, during which Golgi-derived phagophore-like structures appear to engulf proinsulin granules to counteract the accumulation of secretory proteins in the vicinity of the Golgi (Yamaguchi et al., 2016).

Interestingly, the small GTPase Rab2 is required for proper crinophagic degradation of mSGs in Drosophila. In mammals, however, the Rab2 ortholog RAB2A also acts much earlier in the ER–Golgi intermediate compartment (ERGIC) to promote either further maturation or ERAD-mediated degradation of proinsulin, which points to a broader role for RAB2 proteins (herein referring to RAB2A and RAB2B) in regulating the RSP (Sugawara et al., 2014).

Recent studies have found a baseline level of nascent SG degradation even under normal physiological conditions, and they have also shed light on the molecular basis of this process. Overexpression of Rab-interacting lysosomal protein (RILP) enhances the crinophagic elimination of proinsulin in a manner dependent on RAB7 (herein referring to RAB7A and RAB7B) by interacting with SG-localized RAB26, leading to a concomitant decrease in insulin secretion (Zhou et al., 2020). RILP is also known to act as a RAB12 effector in dynein-mediated retrograde transport of mast cell SGs along microtubules (Efergan et al., 2016). Thus, the compromised insulin release upon RILP overexpression could be caused by both the enhanced depletion of insulin granules and their increased retrograde transport (Zhou et al., 2020). In line with this, Drosophila Rab26 also localizes to smaller iSGs and inhibits granule acidification, as well as the rearrangement of their contents and lysosomal fusion (Boda et al., 2023). Thus, Rab26 presumably keeps granules in an immature state until they reach their proper size and composition; it is then removed from mSGs by the Rab7 activator Mon1 and is replaced by Rab7, which promotes further maturation and acidification (Boda et al., 2023). Recently, vesicle-associated membrane protein 4 (VAMP4) has been reported to regulate insulin levels by forming a SNARE complex with syntaxin 7 (STX7), syntaxin 8 (STX8) and vesicle transport through interaction with t-SNAREs 1B (VTI1B) proteins to mediate fusion between proinsulin granules and lysosomes under basal conditions, as well as the lysosomal targeting of CCVs retrieved from maturing granules (Li et al., 2022). VAMP4 is sorted at the TGN into the membrane of forming SGs, which on one hand directs a significant fraction of these granules towards direct lysosomal breakdown. On the other hand, VAMP4 is removed from the remaining population of nascent granules and coalesces with other non-granule components in CCVs in an AP1- and/or clathrin-mediated manner, eventually also reaching lysosomes (Li et al., 2022).

The preferred release of younger granules over older ones has been observed previously in pulse–chase labeling assays (Gold et al., 1982; Halban, 1982). This observation has recently been confirmed with the use of a fluorescent timer reporter that enables insulin SGs to be separated by age (Yau et al., 2020). Based on this, early control of the amount of immature or younger SGs via crinophagy can profoundly influence how much secretory material is released. Lysosomal hydrolases exit the RSP via CCVs during SG maturation (similar to VAMP4), and subsequent fusion of these CCVs with lysosomes is mediated by the same SNARE complex as in crinophagy (Li et al., 2022). Therefore, early nascent granule degradation and lysosome biogenesis both rely on VAMP4 function.

As mentioned above, crinophagy of mSGs is observed in a wide variety of both endocrine and exocrine secretory cell types, especially upon inhibition of secretion. Initial characterization at the ultrastructural level (Creutzfeldt et al., 1969; Kamijo et al., 1986; Koike et al., 1982; Orci et al., 1968, 1984; Smith and Farquhar, 1966) found that mSGs directly fuse with acid phosphatase-positive lysosomal structures, forming multigranular bodies with structures resembling granule dense cores (Orci et al., 1984; Smith and Farquhar, 1966). The presence of insulin in crinophagic bodies has been confirmed in β-cells (Orci et al., 1984), and this insulin is only slowly degraded owing to its crystalline state, in contrast to proinsulin or C-peptide, which are rapidly broken down (Halban et al., 1987). The most pronounced intracellular insulin degradation occurs at low or moderate glucose concentrations, as has been observed by using radioactively labeled leucine (Halban and Wollheim, 1980). Consistent with this, insulin biogenesis starts at a lower glucose concentration than its secretion, resulting in excess SGs, which are removed by crinophagy (Ashcroft, 1980; Schnell et al., 1988). Therefore, crinophagy is activated at glucose concentrations that cause a production of insulin-containing SGs that exceeds the secretory capacity of the cell (Schnell and Borg, 1985; Schnell et al., 1988). Based on this, β-cells appear biased towards insulin biogenesis and modulate their insulin stores by continuous crinophagy of excess SGs beyond necessary secretory demand. This ensures that the SG pool contains mostly younger granules and can be quickly replenished after secretion.

Insulin granules accumulating in isolated pancreatic islet cells of secretion-deficient RAB3A-mutant mice are also eliminated by macro- and micro-secretophagy in addition to crinophagy (Marsh et al., 2007). Since the expression levels of Atg genes is unchanged in the mutants, microsecretophagy is believed to be a major contributor to the observed SG degradation (Marsh et al., 2007). Normally, macrosecretophagy is clearly involved in lysosomal degradation of insulin SGs, as proinsulin can be detected in autophagosomes (Riahi et al., 2016). Inhibition of macroautophagy by double knockdown of Atg5 and Atg7 or by treatment with bafilomycin A1 (a V-ATPase inhibitor) increases both proinsulin content and insulin secretion, and aggregates positive for the autophagy receptor p62 and proinsulin are observed in Atg7-knockout β-cells due to a block of their autophagic degradation (Riahi et al., 2016). The increased insulin release in this case can be explained by prolonged retention of proinsulin in the RSP. In contrast, activation of macroautophagy with Tat–Beclin-1 treatment leads to a decrease intracellular proinsulin levels (Riahi et al., 2016). Becn1F121A mutation-induced hyperactivation of autophagy has been observed to have different effects on insulin-producing and insulin-responsive cells. In β-cells, autophagosomes sequester insulin granules to mediate their lysosomal degradation, whereas in peripheral cells, the induction of autophagy increases insulin responsiveness by alleviating ER stress (Yamamoto et al., 2018). Based on these observations, we suggest that pancreatic β-cells (and possibly other secretory cell types) control the age of their SG pool by continuous removal of old, damaged or obsolete granules via direct SG–lysosome fusion, and by micro- and macro-secretophagy, although the balance and importance of these parallel degradative pathways is unclear. We note that microsecretophagic degradation is only feasible for smaller SGs such as insulin granules. A potential complication in the interpretation of loss-of-function studies is that macroautophagy also appears to be essential for proper β-cell function: a lack of Atg7 leads to a reduction in β-cell mass and impaired insulin secretion (Ebato et al., 2008; Jung et al., 2008).

Crinophagic degradation of insulin granules depends on the level of the mediator prostaglandin E2 (PGE2). PGE2 is produced by cyclooxygenase-2 (COX-2, also known as PTGS2), the activity of which is maintained by nitric oxide that is generated by constitutive activity of neuronal and endothelial nitric oxide synthase (nNOS and eNOS, also known as NOS1 and NOS3, respectively) (Sandberg and Borg, 2006). Furthermore, interleukin 1β enhances crinophagy by stimulating inducible nitric oxide synthase (iNOS, also known as NOS2), but inhibition of iNOS only decreases insulin degradation to a basal level as constitutive nitric oxide synthase activity is preserved (Sandberg and Borg, 2006). Steroid hormones also regulate crinophagy: progesterone activates and corticosterone inhibits the lysosomal fusion of insulin granules via the same glucocorticoid receptor (Sandberg and Borg, 2007). Progesterone has been suggested to promote COX-2 expression by activating nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) (Sandberg and Borg, 2007). Estrogen induces crinophagic degradation of LTH granules in lactotrophs of the anterior pituitary, but since estrogen also induces synthesis and secretion of LTH, increased crinophagy might be a secondary effect of elevated LTH biosynthesis (Kuriakose et al., 1989; Weckman et al., 2014). Steroid (ecdysone) receptors also regulate the acidification and maturation of SGs in Drosophila salivary gland cells via regulation of V-ATPase activity (Nagy et al., 2022). These results suggest that steroid signaling may be a common regulator of SG maturation and crinophagy in most types of secretory cells.

The molecular details of crinophagic mSG degradation have so far only been elucidated for mucin-containing glue granules of Drosophila salivary gland cells, as discussed below (Fig. 2). In human cells, loss of vacuolar protein sorting 41 (VPS41), a subunit of the HOPS tethering complex, impairs the proper maturation and secretion of insulin SGs, presumably by affecting the retrograde pathway, but this effect has been suggested to be independent of the HOPS complex (Burns et al., 2021). Interestingly, VPS41 can self-assemble to form a coat that promotes SG budding from the TGN in neuroendocrine PC-12 cells, so VPS41 might regulate insulin SG maturation in this way (Asensio et al., 2013).

Contrary to an earlier model proposing that crinophagy only removes excess, damaged or obsolete SGs (Csizmadia and Juhász, 2020; Koike et al., 1982; Smith and Farquhar, 1966), more recent findings have shown that crinophagy can also degrade iSGs that have just been shed from the TGN (Csizmadia et al., 2022; Goginashvili et al., 2015; Li et al., 2022; Pasquier et al., 2019; Rajak et al., 2021; Zhou et al., 2020). Since defective SG biogenesis and maturation, which presumably results in abnormal SGs, leads to early activation of crinophagy (Csizmadia et al., 2022; Gehart et al., 2012; Hummer et al., 2017; Hussain et al., 2018; Zhang et al., 2017), cells can probably somehow sense the quality (proper membrane or content composition, size, etc.) of SGs and promote crinophagy of SGs that are improperly formed or damaged. This quality control is particularly important for younger granules, which are likely to be preferentially secreted, as has been shown for insulin SGs (Gold et al., 1982; Halban, 1982; Yau et al., 2020), whereas older granules are presumably sent for degradation.

What are the quality signals that determine whether a particular granule is secreted or degraded? Both degradative and rescue signals can exist on the surface of SGs. Ubiquitin might play a role in designating granules for degradation (Csizmadia and Lőw, 2020), whereas more efficient binding of younger SGs to microtubules and their facilitated transport along microtubule tracks towards the plasma membrane can account for their preferential release (Hu et al., 2021) as they evade lysosomal degradation. Myosin-mediated transport of SGs along filamentous actin to the plasma membrane (Brozzi et al., 2012), as well as recruitment of RAB3 (herein referring to RAB3 isoforms in general) and RAB27A small GTPases, are essential for efficient SG secretion (Cazares et al., 2014; Yi et al., 2002), so it is possible that younger SGs acquire these molecular determinants more efficiently or earlier than older SGs.

Membrane fusion events are controlled by the coordinated action of small GTPases, tethering factors and SNARE proteins (Fig. 4). Rab GTPases determine membrane identity and also recruit effector proteins, such as motor adaptors to promote the transport of the particular vesicle, and tethers to dock vesicles (Stenmark, 2009; Takáts et al., 2018a). The heterohexameric HOPS tethering complex can simultaneously interact with two small GTPase proteins (or their adaptors such as PLEKHM1) via its VPS39 and VPS41 subunits at opposite ends, thus acting as a tether for membranes that contain RAB2, ARL8 or RAB7, such as autophagosomes, late endosomes and lysosomes (Balderhaar and Ungermann, 2013; Lőrincz and Juhász, 2020; Schleinitz et al., 2023). Membrane fusion requires proper assembly of SNAREs. Depending on the central amino acid in the so-called zero layer of assembled SNARE complexes, SNAREs are subdivided into Q-SNAREs and R-SNAREs; a fusion-compatible complex is always formed through the interaction of three Q-SNAREs (Qa, Qb and Qc) and one R-SNARE (Jahn and Scheller, 2006).

Fusion between iSGs and lysosomes relies on the coordinated action of SG-localized RAB26 and lysosomal RAB7 with its effector RILP and a SNARE complex consisting of STX7 (Qa-SNARE), VTI1B (Qb-SNARE), STX8 (Qc-SNARE) and VAMP4 (R-SNARE) (Boda et al., 2023; Li et al., 2022; Zhou et al., 2020). This SNARE complex is similar to the complex between STX7, VTI1B, STX8 and either VAMP7 or VAMP8 that mediates heterotypic fusion of late endosomes with lysosomes that contain VAMP7 (Pryor et al., 2004) or homotypic fusion with other late endosomes if these contain VAMP8 (Antonin et al., 2000a,b; Pryor et al., 2004). VAMP4 is mainly localized to the TGN (Tran et al., 2007) and is involved in retrograde transport from early and/or recycling endosomes to the TGN, where it forms a complex with STX16 (Qa-SNARE), VTI1A (Qb-SNARE) and STX6 (Qc-SNARE) (Mallard et al., 2002). VAMP4 is also found on iSGs, where it mediates the degradation of some nascent SGs by crinophagy, and has been shown to be removed from maturing granules in a clathrin-mediated manner, thereby promoting lysosomal fusion of the resulting CCVs by the same SNARE complex (Li et al., 2022). This reinforces the concept that the membrane composition of SGs is extensively remodeled during their maturation.

During lysosomal fusion of mature glue granules in Drosophila, the small GTPases Rab2, Rab7 and Arl8; the tethering complex HOPS; and the Syntaxin 13 (Syx13; a Qa-SNARE), SNAP29 (which comprises a Qb- and Qc-SNARE motif) and VAMP7 (R-SNARE) complex are all important (Boda et al., 2019; Csizmadia et al., 2018; Lőrincz et al., 2019). This resembles autophagosome–lysosome fusion in Drosophila and mammals, which depends on the same GTPases and tether, and on similar SNARE complexes (Boda et al., 2019; Gutierrez et al., 2004; Hegedűs et al., 2016; Itakura et al., 2012; Jiang et al., 2014; Lőrincz et al., 2017; Takáts et al., 2013, 2014). The R-SNARE YKT6 also plays a role in this fusion event (Bas et al., 2018; Matsui et al., 2018; Takáts et al., 2018b), potentially by either forming a pre-fusion complex involved in HOPS recruitment (Takáts et al., 2018b) or an alternative SNARE complex with endosomal syntaxin 7 (Matsui et al., 2018) (Fig. 4). In Drosophila, overexpression of Vps8 – a subunit of the early endosomal tether class C core vacuole/endosome tethering (CORVET) complex – has been shown to inhibit HOPS assembly by outcompeting Vps41, resulting in inhibition of all HOPS-dependent fusion events, including crinophagy (Lőrincz et al., 2019).

The differences between STX7–VTI1B–STX8–VAMP4 and Syx13–SNAP29–VAMP7 complexes that have been proposed to play a role in crinophagy can be explained by differences in either the examined secretory cell types or SG maturation status. Since VAMP4 is removed from SGs during their maturation (Li et al., 2022), we hypothesize that the lysosomal fusion of mature insulin granules likely relies on a different SNARE complex, whose composition might be similar to the SNARE complex comprising Syx13, SNAP29 and VAMP7 identified in Drosophila (Csizmadia et al., 2018). Further studies are necessary to clarify the role of these SNAREs in mammalian crinophagy of mSGs. Conversely, as disruption of retrograde trafficking triggers early degradation of immature glue granules (Csizmadia et al., 2022), the Syx16-deficient (retrograde transport-defective) genetic background would be suitable to test the role of specific SNAREs involved in iSG degradation in Drosophila, including those identified in mammals (Li et al., 2022).

Contrary to the initial assumption that crinophagy degrades mainly old, abnormal and excess SGs, it has become evident that this process can control both the quality and quantity of nascent, immature and younger SGs. Therefore, crinophagy is a key regulator of the secretory pathway at two levels. First, it eliminates erroneously formed iSGs. Second, it also controls how much secretory material is released by degradation of not only old or residual granules, but also the pool of freshly synthesized SGs that are secreted preferentially. During metabolic stress, crinophagy plays a protective role in secretory cells by providing nutrients for their survival. Since autophagic degradation of SGs can occur by macrosecretophagy, microsecretophagy and crinophagy pathways, the interplay between these pathways is certainly worth investigating in more detail. Recently, steroid hormones and mTORC1 have been shown to induce crinophagy, but the detailed regulation of the process is not yet fully understood. The mechanisms underlying the decision of whether the fate of a particular SG is degradation, maturation or release are still elusive. Recently, several key molecular factors that act in direct lysosomal fusions of both iSGs and mSGs have been revealed. This paves the way for future development of therapies that selectively target this process to potentially prevent and/or cure a range of metabolic and endocrine diseases (see Box 1).

Elements of all figures were created with Biorender.com.

Funding

The authors' work in this area is supported by the National Research, Development and Innovation Office of Hungary (Biotechnology National Laboratory 2022-2.1.1-NL-2022-00008 and KKP129797 to G.J., ÚNKP-22-5-ELTE-826 and PD135447 to T.C.), the Eötvös Loránd Research Network (Featured Topics grant to G.J.), and the Magyar Tudományos Akadémia (BO/00023/21/8 to T.C.).

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RILP restricts insulin secretion through mediating lysosomal degradation of proinsulin
.
Diabetes
69
,
67
-
82
.

Competing interests

The authors declare no competing or financial interests.